Human respiratory syncytial virus (RSV) and human metapneumovirus (HMPV) are cytoplasmic enveloped RNA viruses of the paramyxovirus family, pneumovirus genus. Their genomes are single strands of negative-sense RNA of 15.2 kb (RSV) or 13.3 kb (HMPV) that encode 10 mRNAs and 11 unique proteins (RSV) or 8 mRNAs and 9 unique proteins (HMPV). Each virus encodes a nucleoprotein N, phosphoprotein P, matrix protein M, small hydrophobic protein SH, major glycoprotein G, fusion glycoprotein F, polymerase factors M2-1 and M2-2, and the polymerase protein L. In addition, RSV encodes two nonstructural proteins NS1 and NS2. Pneumonia virus of mice (PVM) is a close relative of RSV whose natural host is the mouse and which provides a convenient permissive animal model. In last years report, we described the strategy of codon-pair deoptimization (CPD) as a means of developing genetically and phenotypically stable attenuated RSV strains. We continue to characterize these RSV strains. We also are presently completing studies in which the same strategy was applied to PVM, which provides an opportunity to observe the effects of this strategy in a fully permissive host. These RSV and HMPV studies will be described in next years report. Another ongoing project of the past several years has been to (i) investigate possible means of increasing the immunogenicity of RSV vaccine candidates that are based on deletion of the M2-2 ORF or the NS2 gene, and (ii) development of further candidates containing either of these two deletions combined with various other mutations, to provide graded differences in the level of attenuation. This project is still in progress and will be reported next year. We collaborated with Dr. Jeffrey Gorman, QIMR Berghofer Medical Research Institute, Queensland, Australia, to perform a proteomic screen of host cell protein expression in response to wild type RSV. This involved analysis of multiple independent biological replicates of RSV-infected and mock-infected lysates of human airway epithelial A549 cells by two different proteomic strategies. One strategy involved fractionation of lysates by in-solution protein isoelectric focusing (IEF), after which individual fractions were digested using trypsin, subjected to capillary high performance liquid chromatography, and analyzed by tandem mass spectrometry (MS/MS). A second strategy involved analysis of tryptic digests of the unfractionated cellular lysate by an improved MS/MS platform. The two strategies detected the known RSV proteins exclusively in lysates of infected cells, as expected, and in the relative abundances anticipated from previous studies. Unprecedented numbers (3247 - 5010) of host cell protein groups also were quantified, and the infection-specific regulation of a large number (191) of these protein groups was evident based on a stringent false discovery rate cut-off (<1%). Bioinformatic analyses revealed that most of the regulated proteins were potentially regulated by antiviral response pathways involving type I, II, and III interferon, TNF-a, and noncanonical NF-kB2. The regulation of specific protein groups by infection was validated by quantitative Western blotting, and the association of specific protein responses with particular cytokines or response pathways was confirmed by comparable analyses of cytokine-treated A549 cells. This produced the most comprehensive proteomic characterization of host cell responses to RSV available to date. We are presently characterizing responses to RSV mutants, such as mutants lacking the NS1 and/or NS2 accessory protein genes. We also contributed to a large collaborative study, directed by Dr. Fernando Polack (Department of Pediatrics, Vanderbilt University), that investigated factors involved in severe RSV disease in infancy. This large multi-center study evaluated the interaction between TLR4 and environmental factors in RSV disease and identified immune mediators associated with severe illness. Evaluation of infants with RSV bronchiolitis revealed that disease severity was strongly influenced by the TLR4 genotype of the individual and by environmental exposure to lipopolysaccharide (LPS), a TLR4 agonist. RSV-infected infants with severe disease exhibited a high ratio of GATA3 (Th2 master regulator): T-bet (Th1 master regulator), which was associated with a high IL-4/IFN-g ratio in respiratory secretions. This linked severe RSV disease with Th2 polarization. Interestingly, high Th2 polarization was associated with low environmental LPS and with polymorphisms in TLR4 associated with hypo-responsiveness. This is consistent with the expectation that low activation of TLR4 contributes to a Th2 bias. Murine models of RSV infection confirmed that LPS exposure, TLR4 genotype, and Th2 polarization influence disease phenotypes. Together, the results of this study identify environmental and genetic factors that influence RSV pathogenesis and reveal that a high IL-4/IFN-g ratio is associated with severe disease. This identifies potential targets for therapeutic intervention. We have been collaborating with Dr. Peter Kwong of the Vaccine Research Center to develop improved subunit vaccines for the HMPV F protein. These studies are in progress and will be reported next year.